Project Summary Failure to adequately treat pain accounts for hundreds of billions of dollars of lost productivity and medical expenses annually. According to the Centers for Disease Control, each day in the United States over forty people die from an overdose of prescription pain killers (e.g. Vicodin and OxyContin). Consequently, there is an urgent need to develop new, safe, and potent non-opioid analgesics for the treatment of acute and chronic pain. Many surgical procedures induce significant acute pain that is difficult to treat. Patients who undergo such major surgical procedures are also at an increased risk of developing a subsequent opioid addiction. Therefore, improving acute pain control will not only enhance patient outcomes but may also lead to reduced prevalence of subsequent opioid abuse. The endocannabinoid 2-arachidonoylglycerol (2-AG) produces analgesia by activating cannabinoid receptors. However, 2-AG can also be hydrolyzed by the enzyme monoacylglycerol lipase (MAGL) to generate arachidonic acid, the precursor to downstream eicosanoids that can promote pain. In a recent publication, our group demonstrated that 2-AG levels were elevated in patients who developed greater acute postoperative pain, suggesting that 2-AG/eicosanoid crosstalk may directly modulate acute pain in humans. However, the contribution of 2-AG metabolism toward acute pain is poorly defined and its role in eicosanoid biosynthesis and pain in humans is lacking, highlighting a major gap in our understanding of endocannabinoid metabolism and pain. The current proposal leverages rodent surgical models and patient derived samples to test the major hypothesis that MAGL activity is essential for the biosynthesis of cyclooxygenase and 5-lipoxygenase (5-LOX) derived eicosanoids, which we hypothesize operate in parallel to promote acute pain. In Aim 1, we will employ complementary pharmacological and genetic approaches to test the hypothesis that MAGL inhibition suppresses acute pain by depriving cyclooxygenase and 5-LOX enzymes of arachidonic acid for eicosanoid biosynthesis within the incision site. This aim will also employ selective inhibitors and 5-LOX KO mice to test the hypothesis that 5-LOX inhibition attenuates acute pain. Aim 2 will leverage novel conditional MAGL knockout mice to identify peripheral cell populations wherein MAGL activity contributes to postoperative eicosanoid biosynthesis and pain. Aim 3 will characterize 2-AG/eicosanoid crosstalk in perioperative human tissue and will assess the contribution of 2-AG and eicosanoid levels toward acute pain in humans. The outcome of this study will provide fundamental insights into endocannabinoid/eicosanoid crosstalk and may identify MAGL as a novel target for the treatment of acute pain, thereby providing the foundation for the rapid translation of MAGL inhibitors to patients suffering from inadequately controlled pain.